1. Field of the Invention
The present invention is related to the fields of molecular biology, virology, immunology and medicine. The invention provides a composition comprising an ordered and repetitive antigen or antigenic determinant array, and in particular a RANKL protein, RANKL fragment or RANKL peptide-array. More specifically, the invention provides a composition comprising a virus-like particle and at least one RANKL protein, RANKL fragment or RANKL peptide bound thereto. The invention also provides a process for producing the conjugates and the ordered and repetitive arrays, respectively. The compositions of the invention are useful in the production of vaccines for the treatment of bone diseases and as a pharmaccine to prevent or cure bone diseases and to efficiently induce immune responses, in particular antibody responses. Furthermore, the compositions of the invention are particularly useful to efficiently induce self-specific immune responses within the indicated context.
2. Related Art
Living bone is permanently turned over by balanced and coordinated remodeling processes. Primarily two cell types contribute to this remodeling: osteoblasts are essential for the formation of bone while osteoclasts promote dissolution of bone matrix and solubilization of the hydroxyapatite. In young individuals with growing bone, the rate of bone formation exceeds the rate of bone resorption, while in older individuals the rate of resorption can exceed formation and result in a net loss of bone mineral density and/or bone mass. In the latter case the bone strength is weakened and leads to an increased risk of fracture as well as slow or incomplete repair of broken bones. Multiple conditions in humans are known to be associated with an imbalance in bone remodeling.
Recently three proteins have been described that are crucially involved in the formation of osteoclasts from hematopoietic precursor cells and regulation of bone remodeling. RANKL (Receptor activator of NFkB Ligand) which is also known as TNFSF11 (Tumor necrosis factor superfamily member 11), TRANCE (TNF-related activation induced cytokine), ODF (Osteoclast differentiation factor) or OPGL (Osteoprotegerin ligand) is a transmembrane protein of 245 amino acids that forms homotrimers. Part of the extracellular region of RANKL can be shed by a TACE-like protease. In addition, splice variants lacking the transmembrane region have been described. The shed part of RANKL contains the domain that is highly homologous to the TNF-α (Lum, L., et al., J. Biol. Chem. 274: 13613-13618 (2000)).
Processes how to produce RANKL protein and RANKL fragments have been disclosed in WO 9846751, U.S. Pat. No. 5,843,678, WO 98259958, U.S. Pat. No. 6,242,586, WO 9828426, U.S. Pat. No. 6,242,213, WO 9929865, JP 2000102390 and WO 0015807.
RANKL interacts with a transmembrane molecule on osteoclasts, termed RANK (Receptor activator of NFkB). This interaction leads to activation of the osteoclast precursor and ends in the formation of active, bone-resorbing osteoclasts. In vivo, a soluble decoy receptor termed osteoprotegerin, is involved in the regulation of osteoclastogenesis by its ability to bind to RANKL and inhibit the interaction of RANKL with its receptor RANK. This inhibition leads to a suppression of osteoclastogenesis and thus provides a means to stop excessive bone resorption. The interaction of RANKL with its receptor RANK can be suppressed by recombinant osteoprotegerin and by a soluble RANK-Fc fusion protein. In accordance with these findings, RANKL- and RANK-deficient mice develop osteopetrosis while RANKL-overexpressing transgenic mice as well as osteoprotegerin-deficient mice develop osteoporosis (Kong Y Y., et al., Nature 397:315-322 (1999), Kim, N., et al., Proc. Natl. Acad. Sci USA 97:10905-10910 (2000), Dougall, B., et al., Proc. Natl. Acad. Sci USA 97:1566-1571 (1999), Bucay, N., et al., Genes Dev. 12: 1260-1268 (1998)).
The importance of the RANKL-RANK-osteoprotegerin system is further confirmed in an rodent model for osteoporosis induced by estrogen-deficiency. Recombinant osteoprotegerin completely abolished ovariectomy-induced bone loss (Simonet, W. S., et al. Cell 89:309-319 (1997).
In an adjuvant-induced arthritis model osteoprotegerin injection was able to prevent bone loss and cartilage destruction, but not inflammation (paw swelling). Beside its expression on stromal cells RANKL is also expressed on T cells, and RANK is found on antigen-presenting cells. It is assumed that during an arthritic reaction activated T cells with enhanced RANKL expression mediate an increase in osteoclastogenesis and subsequent bone loss. The interaction of RANKL with RANK also enhances the longevity and adjuvant properties of dendritic cells (Kong Y. Y., et al., Nature 402:304-309 (1999)).
Alveolar bone destruction and subsequent tooth loss is observed in periodontal infections. In vivo inhibition of RANKL function with osteoprotegerin diminished alveolar bone destruction and reduced the number of periodontal osteoclasts after microbial challenge (Teng, Y. T. A., et al., J. Clin. Invest. 106:R59-R67 (2000).
Bone tumors and certain tumor metastases are characterized by increased bone resorption due to an increased osteoclastogenesis (Hofbauer, L. C. and Heufelder A. E., J. Clin Endocrin. Met. 85:2355-2363 (2000). Osteoprotegerin was shown to inhibit prostate-cancer induced osteoclastogenesis and prevent prostate tumor growth in the bone of mice (Zhang Y., et al., J. Clin. Invest. 107:1219-1220 (2001). It also diminished advanced bone cancer pain in mice (Luger N. M., et al., Cancer Res. 61:4038-4047 (2001)). Multiple myeloma is a B cell malignancy characterized by the accumulation of plasma cells in the bone marrow and the development of osteolytic bone disease. In mouse models for multiple myeloma injection of osteoprotegerin or RANK-Fc fusion protein prevented the development of lytic bone lesions and interfered with myeloma progression (Pearse R N., et al., Proc. Natl. Acad. Sci USA 98:11581-11586 (2001).
Central to the etiology of aseptic loosening of prosthetic implants is periprostethic osteolysis at the bone-implant interface, which is caused by wear-debris-induced inflammation. Fibroblast-like synoviocytes, transfected with osteoprotegerin, were able to prevent wear debris induced osteoclastogenesis in a mouse model (Gouter J. J., et al., J. Orthop. Res. 202:169-173 (2002)).
Vascular calcification is found with high clinical incidence in the osteoporotic patient population. An involvement of the RANKL-RANK-osteoprotegerin system is demonstrated by the finding that osteoprotegerin-deficient mice showed arterial calcification which could be reversed by recombinant osteoprotegerin (Min, H., et al., J. Exp. Med. 192:463-474 (2000)).
All these finding point to a crucial importance of the RANKL-RANK-osteoprotegerin system in regulation bone resorption in a variety of pathological conditions. So far, inhibition of bone loss has been mainly shown by injection of recombinant osteoprotegerin or a RANK-Fc fusion protein. Conceptually, immunization of an animal with RANKL should allow the production of RANKL-specific antibodies which, by binding to the RANK binding site or steric inhibition, should interfere with osteoclastogenesis.
However, so far no vaccination with a RANKL protein or peptide has been reported. Moreover, there has been no evidence that vaccines might be effective for protection against bone diseases, in particular, since it is usually difficult to induce antibody responses to self-molecules by conventional vaccination.
One way to improve the efficiency of vaccination is to increase the degree of repetitiveness of the antigen applied. Unlike isolated proteins, viruses induce prompt and efficient immune responses in the absence of any adjuvants both with and without T-cell help (Bachmann and Zinkernagel, Ann. Rev. Immunol: 15:235-270 (1991)). Although viruses often consist of few proteins, they are able to trigger much stronger immune responses than their isolated components. For B-cell responses, it is known that one crucial factor for the immunogenicity of viruses is the repetitiveness and order of surface epitopes. Many viruses exhibit a quasi-crystalline surface that displays a regular array of epitopes which efficiently crosslinks epitope-specific immunoglobulins on B cells (Bachmann and Zinkernagel, Immunol. Today 17:553-558 (1996)). This crosslinking of surface immunoglobulins on B cells is a strong activation signal that directly induces cell-cycle progression and the production of lgM antibodies. Further, such triggered B cells are able to activate T helper cells, which in turn induce a switch from IgM to IgG antibody production in B cells and the generation of long-lived B cell memory—the goal of any vaccination (Bachmann and Zinkernagel, Ann. Rev. Immunol. 15:235-270 (1997)). Viral structure is even linked to the generation of anti-antibodies in autoimmune disease and as a part of the natural response to pathogens (see Fehr, T., et al., J Exp. Med. 185:1785-1792 (1997)). Thus, antibodies presented by a highly organized viral surface are able to induce strong anti-antibody responses.
As indicated, however, the immune system usually fails to produce antibodies against self-derived structures. For soluble antigens present at low concentrations, this is due to tolerance at the Th cell level. Under these conditions, coupling the self-antigen to a carrier that can deliver T help may break tolerance. For soluble proteins present at high concentrations or membrane proteins at low concentration, B and Th cells may be tolerant. However, B cell tolerance may be reversible (anergy) and can be broken by administration of the antigen in a highly organized fashion coupled to a foreign carrier (Bachmann and Zinkernagel, Ann. Rev. Immunol. 15:235-270 (1997)).